EFFLUX OF CELLULAR CHOLESTEROL AND PHOSPHOLIPID TO LIPID-FREE APOLIPOPROTEINS AND CLASS-A AMPHIPATHIC PEPTIDES

The mechanism(s) by which lipid-free apolipoprotein (ape) AI is able t o stimulate efflux of cholesterol and phospholipid from cells in cultu res has (have) been examined. This process was found to be enhanced wh en macrophages were enriched with cholesterol. There were 12- and 4-fo ld increases in cholesterol and phospholipid efflux, respectively, fro m cholesterol-enriched mouse macrophages when compared to cells not lo aded with cholesterol. This enhancement in cholesterol efflux to lipid -free apo AI from macrophages enriched with cholesterol was found to b e controlled by the level of free cholesterol in the cells. When chole sterol-enriched mouse macrophages were exposed to lipid-free apo AI at 20 mu g/mL (706 nM), there was significant efflux of [C-14]cholestero l and [H-3]phospholipid (20% +/- 0.5%/24 h and 6% +/- 0.3%/24 h, respe ctively). In comparison, HDL at equivalent protein concentrations only stimulated 11% and 4% efflux of cholesterol and phospholipid, respect ively. Synthetic peptides containing amphipathic helical segments that mimic those present in apo AI were used to examine the structural fea tures of the apoprotein which stimulate lipid efflux. Peptides contain ing only one (18A) or two (37pA) amphipathic helical segments stimulat ed as much cholesterol efflux from both mouse macrophages and L-cells as apo AI. The order of efficiency, as assessed by the mass concentrat ion at which half-maximal efflux was reached (EC(50)), was apo AI > 37 pA > 18A, indicating that acceptor efficiency was dependent on the num ber of amphipathic helical segments per molecule. When the helical con tent of 18A was increased by neutralizing the charges at the ends of t he peptide (Ac-18A-NH2), there was a substantial increase in the effic iency for cholesterol efflux (EC(50) 18A = 17 mu g/mL vs Ac-18A-NH2 = 6 mu g/mL). In contrast, when the amphipathicity of the helix in 18A w as decreased by scrambling the amino acid sequence, thereby reducing i ts lipid affinity, cholesterol and phospholipid efflux were not stimul ated. The efficiency with which the peptides stimulated cholesterol ef flux was in order of their lipid affinity (37pA > Ac-18A-NH2 > 18A), a nd this order was similar for phospholipid efflux. The time courses of lipid release from mouse macrophages and L-cells indicated that phosp holipid appeared in the extracellular medium before cholesterol. These results suggest that the apo AI or peptides first interacted with the cell to form protein/phospholipid complexes, that could then accept c holesterol.